Rice hybrid XL730

ABSTRACT

A rice hybrid designated XL730 is disclosed. The invention relates to the seeds of rice hybrid XL730, to the plants of rice hybrid XL730 and to methods for producing a rice plant produced by crossing the hybrid XL730 with itself or another rice plant. The invention further relates to hybrid rice seeds and plants produced by crossing the hybrid XL730 with another rice plant.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive rice hybridcultivar designated XL730. All publications cited in this applicationare herein incorporated by reference.

Rice is an ancient agricultural crop and is today one of the principalfood crops of the world. There are two cultivated species of rice: Oryzasativa L., the Asian rice, and Oryza glaberrima Steud., the Africanrice. The Asian species constitutes virtually all of the world'scultivated rice and is the species grown in the United States. Threemajor rice producing regions exist in the United States: the MississippiDelta (Arkansas, Mississippi, northeast Louisiana, southeast Missouri),the Gulf Coast (southwest Louisiana, southeast Texas), and the CentralValleys of California.

Rice production in the United States can be broadly categorized aseither dry-seeded or water-seeded. In the dry-seeded system, rice issown into a well-prepared seed bed with a grain drill or by broadcastingthe seed and incorporating it with a disk or harrow. Moisture for seedgermination is from irrigation or rainfall. Another method of plantingby the dry-seeded system is to broadcast the seed by airplane into aflooded field, then promptly drain the water from the field. For thedry-seeded system, when the plants have reached sufficient size (four-to five-leaf stage), a shallow permanent flood of water 5 to 16 cm deepis applied to the field until the rice approaches maturity. Rice isgrown on flooded soils to optimize grain yields. Heavy clay soils orsilt loam soils with hard pan layers about 30 cm below the surface aretypical rice-producing soils because they minimize water losses due topercolation.

In the water-seeded system, rice seed is soaked for 12 to 36 hours toinitiate germination, and the seed is broadcast by airplane into ashallow-flooded field. Water may be drained from the field for a shortperiod of time to enhance seedling establishment or the seedlings may beallowed to emerge through the shallow flood. In either case, a shallowflood is maintained until the rice approaches maturity. For both thedry-seeded and water-seeded production systems, the rice is harvestedwith large combines 2 to 3 weeks after draining.

Rice in the United States is classified into three primary market typesby grain size and shape as: long-grain, medium grain and short-grain.Typical U. S. long-grain rice cooks dry and fluffy when steamed orboiled, whereas medium- and short-grain rice cooks moist and sticky.Long-grain cultivars have been traditionally grown in the southernstates and generally receive higher market prices.

Although specific breeding objectives vary somewhat in the differentregions, increasing yield is a primary objective in all programs. Grainyield of rice is determined by the number of panicles per unit area, thenumber of fertile florets per panicle, and grain weight per fertilefloret. Increases in any or all of these yield components provide amechanism to obtain higher yields. Heritable variation exists for all ofthese components, and breeders may directly or indirectly select forincreases in any of them.

There are numerous steps in the development of any novel, desirablecultivar. Plant breeding begins with the analysis and definition ofproblems and weaknesses of the current cultivars, followed by theestablishment of program goals, and the definition of specific breedingobjectives. The next step is selection of parental lines that possessthe traits required to meet the program goals. The goal is to combine ina single cultivar an improved combination of desirable traits from theparental sources. These important traits may include higher yield,resistance to diseases and insects, better stems and roots, tolerance tolow temperatures, better agronomic characteristics, and grain quality.

The goal of rice plant breeding is to develop new, unique and superiorrice cultivars and hybrids. The breeder initially selects and crossestwo or more parental lines, followed by selection among the many newgenetic combinations. The breeder can theoretically generate billions ofnew and different genetic combinations via crossing. The breeder has nodirect control at the cellular level; therefore, two breeders will neverdevelop the same line, or even very similar lines, having the same ricetraits.

Choice of breeding methods to select for the improved combination oftraits depends on the mode of plant reproduction, the heritability ofthe trait being improved, and the type of cultivar used commercially(e.g., F₁ hybrid cultivar, pureline cultivar, etc.). For highlyheritable traits, a choice of superior individual plants evaluated at asingle location will be effective, whereas for traits with lowheritability, selection should be based on mean values obtained fromreplicated evaluations of families of related plants. Popular selectionmethods include pedigree selection, backcross selection, and single seedselection, or a combination of these methods.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops such as rice. Two parents which possessfavorable, complementary traits are crossed to produce an F₁. An F₂population is produced by selfing one or several F₁s. Selection of thebest individuals may begin in the F₂ population; then, beginning in theF₃, the best individuals in the best families are selected. Replicatedtesting of families can begin in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new parental lines.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, rice breeders commonly harvest one or moreseeds from each plant in a population and thresh them together to form abulk. Part of the bulk is used to plant the next generation and part isput in reserve. The procedure has been referred to as modifiedsingle-seed descent or the pod-bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster to thresh panicles with a machine than to removeone seed from each by hand for the single-seed procedure. Themultiple-seed procedure also makes it possible to plant the same numberof seeds of a population each generation of inbreeding. Enough seeds areharvested to make up for those plants that did not germinate or produceseed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, R. W. et al. Principles of Plant Breeding (1999);Agrawal, R. L. Fundamentals of Plant Breeding and Hybrid Seed Production(1998); Schlegel, R. H. J. Encyclopedic Dictionary of plant Breeding andRelated Subjects (2003); Fehr, W. R. et al. Principles of CultivarDevelopment—Theory and Technique (1987)).

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for at least three or more years. The best lines arecandidates for new commercial cultivars; those still deficient in a fewtraits may be used as parents to produce new populations for furtherselection.

These processes, which lead to the final step of marketing anddistribution, usually take from 8 to 12 years from the time the firstcross is made and may rely on the development of improved breeding linesas precursors. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

Each breeding cycle, the plant breeder selects the germplasm to advanceto the next generation. This germplasm is grown under unique anddifferent geographical, climatic and soil conditions, and furtherselections are then made, throughout the growing season. The cultivarswhich are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same cultivar twice by using theexact same original parents and the same selection techniques. Thisunpredictability results in the expenditure of large amounts of researchmonies to develop superior new rice cultivars.

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Rice, Oryza sativa L., is an important and valuable field crop. Thus, acontinuing goal of rice breeders is to develop stable, high yieldingrice cultivars that are agronomically sound. The reasons for this goalare to maximize the amount of grain produced on the land used and tosupply food for both animals and humans. To accomplish this goal, therice breeder must select and develop rice plants that have the traitsthat result in superior cultivars.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary and illustrative, not limiting in scope. In variousembodiments, one or more of the above-described problems have beenreduced or eliminated, while other embodiments are directed to otherimprovements.

The present invention relates to a hybrid rice cultivar, designatedXL730, and seeds and plants derived from the hybrid. The invention alsorelates to hybrid plants and seeds and any further progeny ordescendants of the hybrid derived by crossing XL730 as a pollen donor.The invention is also directed to methods for producing a rice plant bycrossing a first parent rice plant with a second parent rice plantwherein the first parent rice plant is 054001 and the second parent riceplant is 053004. Thus, any methods using the hybrid rice line XL730 inbackcrosses, hybrid production, crosses to populations, and the like,are part of this invention. All plants which are a progeny of or descendfrom hybrid rice line XL730 are within the scope of this invention. Itis an aspect of this invention for rice hybrid XL730 to be used incrosses with other, different, rice plants to produce first generation(F₁) rice hybrid seeds and plants with superior characteristics.

In another aspect, the present invention provides for single geneconverted plants of the parents of XL730. The single transferred genemay preferably be a dominant or recessive allele. Preferably, the singletransferred gene will confer such traits as herbicide resistance, insectresistance, resistance for bacterial, fungal, or viral disease, malefertility, male sterility, enhanced nutritional quality, and industrialusage. The single gene may be a naturally occurring rice gene or atransgene introduced through genetic engineering techniques.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of rice plant XL730. The tissue culture willpreferably be capable of regenerating plants having the physiologicaland morphological characteristics of the foregoing rice plant, and ofregenerating plants having substantially the same genotype as theforegoing rice plant. Preferably, the regenerable cells in such tissuecultures will be embryos, protoplasts, meristematic cells, callus,pollen, leaves, flowers, anthers, pistils, root tips, glumes, seeds,panicles or stems. Still further, the present invention provides riceplants regenerated from the tissue cultures of the invention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Alkali Spreading Value. Indicator of gelatinization temperature and anindex that measures the extent of disintegration of milled rice kernelin contact with dilute alkali solution. Standard long grains have 3 to 5Alkali Spreading Value (intermediate gelatinization temperature).

Allele. An allele is any of one or more alternative forms of a gene, allof which relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Apparent Amvlose Percent. The most important grain characteristic thatdescribes cooking behavior in each grain class, or type, i.e., long,medium and short grain. It is the amount of amylose in the endospermstarch of milled rice that can be detected using the iodine bindingmethod. Standard long grains contain 20 to 23% amylose. Rexmont typelong grains contain 24 to 25% amylose. Short and medium grains contain16 to 19% amylose. Waxy rice contains 0% amylose. Amylose values willvary over environments.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Breakdown. The peak viscosity minus the trough viscosity.

Chalk. An opaque region of the rice kernel due to loose packing of thestarch granules. Chalk may occur throughout or in a part of the kernel.

Consistency. Final viscosity minus trough viscosity.

Days to 50% heading. Average number of days from emergence to the daywhen 50% of all panicles are exerted at least partially through the leafsheath. A measure of maturity.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the cultivar, except for the characteristics derivedfrom the converted gene.

Final Viscosity. The viscosity measure of rice flour/water slurry afterbeing heated to 95° C. and uniformly cooled to 50° C. (AmericanAssociation of Cereal Chemists). Values less than 200 for cool pasteindicate softer cooking types of rice.

Gelatinization temperature. The temperature at which a definedflour-water mixture exhibits a measurable viscosity increase under astandardized, instrument-specific cooking cycle (also known as “initialviscosity increase temperature”).

Grain Length (L). Length of a rice grain is measured in millimeters.

Grain Width (W). Width of a rice grain is measured in millimeters.

Grain Yield. Grain yield is measured in kilograms per hectare and at12.0% moisture. Grain yield of rice is determined by the number ofpanicles per unit area, the number of fertile florets per panicle, andgrain weight per floret.

Harvest Moisture. The percent of moisture of the grain when harvested.

Length/Width (L/W) Ratio. This ratio is determined by dividing theaverage length (L) by the average width (W).

Lodging Percent. Lodging is measured as a subjective rating and is apercentage of the plant stems leaning or fallen completely to the groundbefore harvest.

1000 Grain Wt. The weight of 1000 rice grains as measured in grams.

Paste Temperature (also called Initial Viscosity Increase Temperature).The temperature at which a defined flour-water mixture exhibits ameasurable viscosity increase under a standardized, instrument-specific(RAPID VISCO Analyzer) cooking cycle.

Paste Time. The time at which a defined flour-water mixture exhibits ameasurable viscosity increase under a standardized, instrument-specific(RAPID VISCO Analyzer) cooking cycle.

Peak Temperature at Peak Viscosity. The temperature at which peak hotpaste viscosity is attained.

Peak Time. The time at which peak (maximum) hot-paste viscosity isattained during a standardized, instrument-specific (RAPID VISCOAnalyzer) cooking cycle.

Plant Height. Plant height in centimeters is taken from soil surface tothe tip of the extended panicle at harvest.

Peak Viscosity. The maximum viscosity attained during heating when astandardized instrument-specific protocol is applied to a defined riceflour-water slurry.

Quantitative Trait Loci (QTL). Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

RVA Viscosity. The RAPID VISCO Analyzer (RVA) is a widely usedlaboratory instrument to examine paste viscosity, or thickening abilityof milled rice during the cooking process.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Setback. Setback is the final viscosity minus trough viscosity.

Single Gene Converted (Conversion). Single gene converted (conversion)plant refers to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a variety are recovered in additionto the single gene transferred into the variety via the backcrossingtechnique or via genetic engineering.

Starch Index. The sum of apparent amylose value plus alkali spreadingvalue. This value correlates with the cooking properties of rice.

Total Milling (also called Milling Yield). The quantity of total milledrice produced in the milling of rough rice to a well-milled degree; itis usually expressed as a percent of rough rice by weight, but whenspecified, may be expressed as a percent of brown rice.

Trough (also known as hot-paste viscosity). Viscosity measure of riceflour/water slurry after being heated to 95° C. Lower values indicatesofter and stickier cooking types of rice.

Trough Time. The time at which the Trough (hot-paste viscosity) occurswhen a defined flour-water mixture has been heated to and held at themaximum temperature of a standardized, instrument-specific (RAPID VISCOAnalyzer) cooking cycle.

Trough Viscosity. The minimum viscosity after the peak, normallyoccurring when the sample starts to cool.

Whole Milling (also called Head Rice Milling Yield). The quantity ofmilled head (¾-whole) rice produced in the milling of rough rice to awell-milled degree, usually expressed in the United States as a percentof rough rice by weight.

DETAILED DESCRIPTION OF THE INVENTION

Rice hybrid XL730 is a high yielding, very early maturing, photoperiodinsensitive, long grain hybrid rice cultivar that is tolerant to theimidazolinone class of herbicides. It is produced from the cross of themale fertile line 053004 and the male sterile line 054001. The hybridhas shown uniformity and stability, as described in the following hybriddescription information. It has been produced and tested a sufficientnumber of years with careful attention to uniformity of plant type. Thehybrid has been increased with continued observation for uniformity ofthe parent lines.

Rice hybrid XL730 has the following morphologic and othercharacteristics (based primarily on data collected at Alvin, Tex.).TABLE 1 HYBRID DESCRIPTION INFORMATION Maturity (Alvin, Texas at 150kg/ha N) Days to maturity: 75 days from emergence to 50% headingMaturity Class: Very early (70-85 days) Culm (Degrees from perpendicularafter flowering) Angle: Erect (less than 30 degrees from vertical)Length: 130 cm (Soil level to top of extended panicle on main stem)Height Class: Tall Internode Color (After flowering): Green Strength(Lodging resistance): Moderately weak (most plants leaning with up to30% lodging) Flag Leaf (After Heading) Length: 36 cm Width: 1.5 cmPubescence: Pubescent Leaf Angle (After heading): Intermediate BladeColor: Green Basal Leaf Sheath Color: Purple Ligule Color (Latevegetative state): Purple lines Shape: Cleft Collar Color (Latevegetative stage): Pale Green Auricle Color (Late vegetative stage):Purple Panicle Length: 22 cm Type: Compact Secondary Branching: LightExsertion (near maturity): 91-99% Axis: Droopy Shattering: Moderate(6-25%) Threshability: Easy Grain (Spikelet) Awns (After full heading):Short and Partly Awned Apiculus Color (At maturity): Purple StigmaColor: Purple Stigma Exsertion (at flowering): 100% Lemma and PaleaColor (At maturity): Straw Lemma and Palea Pubescence: Hairs on upperportion Spikelet Sterility (At maturity): Highly Fertile (>90%) Grain(Seed) Seed Coat Color: Light Brown Endosperm Type: Nonglutinous(nonwaxy) Endosperm Translucency: Clear Endosperm Chalkiness: Small(percentage of grains with more than 20% of chalkiness) Scent:Nonscented Shape Class (Length/width ratio): Long Measurements: Length(mm) Width (mm) L/W Ratio 1000 Grains (grams) Milled 6.79 2.14 3.18 25Milling Yield (% whole kernel 59.2% (head) rice to rough rice): ApparentAmylose: 20.5% Alkali Spreading value: 3.7 (1.5% KOH Solution)Gelatinization Temperature Type: Intermediate Disease Resistance RiceBlast (Pyricularia oryzae): Overall Resistance = Moderately ResistantStraight Head: Moderately Resistant Sheath Blight (Rhizoctonia solani):Moderately Susceptible

This invention is also directed to methods for producing a rice plant bycrossing a first parent rice plant with a second parent rice plant,wherein the first or second rice plant is a rice plant from the hybridXL730. Further, both first and second parent rice plants may be from thehybrid XL730. Therefore, any methods using the hybrid XL730 are part ofthis invention: selfing, backcrosses, hybrid breeding, and crosses topopulations. Any plants produced using rice hybrid XL730 as a parent arewithin the scope of this invention.

Still further, this invention also is directed to methods for producinga rice hybrid XL730-derived rice plant by crossing rice hybrid XL730with a second rice plant and growing the progeny seed, and repeating thecrossing and growing steps with the rice hybrid XL730-derived plant from0 to 7 times. Thus, any such methods using the rice hybrid XL730 arepart of this invention: selfing, backcrosses, hybrid production, crossesto populations, and the like. All plants produced using rice hybridXL730 as a parent are within the scope of this invention, includingplants derived from rice hybrid XL730.

It should be understood that the parents of hybrid XL730 can, throughroutine manipulation of cytoplasmic or other factors, be produced in amale-sterile form. Such embodiments are also contemplated within thescope of the present claims.

Further embodiments of the Invention

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years, several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the parents of the claimed hybrid.

Culture for expressing desired structural genes and cultured cells areknown in the art. Also as known in the art, rice is transformable andregenerable such that whole plants containing and expressing desiredgenes under regulatory control may be obtained. General descriptions ofplant expression vectors and reporter genes and transformation protocolscan be found in Gruber, et al., “Vectors for Plant Transformation”, inMethods in Plant Molecular Biology & Biotechnology, Glich, et al., (Eds.pp. 89-119, CRC Press, 1993). Moreover GUS expression vectors and GUSgene cassettes are available from Clone Tech Laboratories, Inc., PaloAlto, Calif. while luciferase expression vectors and luciferase genecassettes are available from ProMega Corp. (Madison, Wis.). Generalmethods of culturing plant tissues are provided for example by Maki, etal., “Procedures for Introducing Foreign DNA into Plants” in Methods inPlant Molecular Biology & Biotechnology, Glich, et al., (Eds. pp. 67-88CRC Press, 1993); and by Phillips, et al., “Cell-Tissue Culture andIn-Vitro Manipulation” in Corn & Corn Improvement, 3rd Edition; Sprague,et al., (Eds. pp. 345-387) American Society of Agronomy Inc., 1988.Methods of introducing expression vectors into plant tissue include thedirect infection or co-cultivation of plant cells with Agrobacteriumtumefaciens, Horsch et al., Science, 227:1229 (1985). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer provided by Gruber, et al., supra.

Useful methods include but are not limited to expression vectorsintroduced into plant tissues using a direct gene transfer method suchas microprojectile-mediated delivery, DNA injection, electroporation andthe like. More preferably expression vectors are introduced into planttissues using a microprojectile media delivery system with a biolisticdevice or using Agrobacterium-mediated transformation. Transformantplants obtained with the protoplasm of the invention are intended to bewithin the scope of this invention.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed rice plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the rice plant(s).

Expression Vectors for Rice Transformation—Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80:4803 (1983). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant. Hayford et al., PlantPhysiol. 86:1216 (1988); Jones et al., Mol. Gen. Genet., 210:86 (1987);Svab et al., Plant Mol. Biol. 14:197 (1990); Hille et al., Plant Mol.Biol. 7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil. Comai et al.,Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618(1990) and Stalker et al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation not of bacterialorigin include, for example, mouse dihydrofolate reductase, plant5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987), Shahet al., Science 233:478 (1986), Charest et al., Plant Cell Rep. 8:643(1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase and chloramphenicol acetyltransferase.Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBOJ. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987), DeBlock et al., EMBO J. 3:1681 (1984). Another approach to theidentification of relatively rare transformation events has been use ofa gene that encodes a dominant constitutive regulator of the Zea maysanthocyanin pigmentation pathway. Ludwig et al., Science 247:449 (1990).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are available. Molecular Probes publication2908, IMAGENE GREEN, p. 1-4 (1993) and Naleway et al., J. Cell Biol.115:151a (1991). However, these in vivo methods for visualizing GUSactivity have not proven useful for recovery of transformed cellsbecause of low sensitivity, high fluorescent backgrounds and limitationsassociated with the use of luciferase genes as selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Rice Transformation—Promoters

Genes included in expression vectors must be driven by nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters—An inducible promoter is operably linked to agene for expression in rice. Optionally, the inducible promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in rice. With aninducible promoter the rate of transcription increases in response to aninducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Meft et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991)). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone (Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991)).

B. Constitutive Promoters—A constitutive promoter is operably linked toa gene for expression in rice or the constitutive promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in rice.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)).

The ALS promoter, Xba1/Ncol fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said Xba1/Ncolfragment), represents a particularly useful constitutive promoter. SeePCT application WO96/30530.

C. Tissue-specific or Tissue-preferred Promoters—A tissue-specificpromoter is operably linked to a gene for expression in rice.Optionally, the tissue-specific promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in rice. Plants transformed with a gene ofinterest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al., PlantMol. Biol. 9:3-17 (1987); Lerner et al., Plant Physiol. 91:124-129(1989); Fontes et al., Plant Cell 3:483-496 (1991); Matsuoka et al.,Proc. Natl. Acad. Sci. 88:834 (1991); Gould et al., J. Cell. Biol.108:1657 (1989); Creissen et al., Plant J. 2:129 (1991); Kalderon, etal., Cell 39:499-509 (1984); Steifel, et al., Plant Cell 2:785-793(1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is rice. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RFLP, PCR andSSR analysis, which identifies the approximate chromosomal location ofthe integrated DNA molecule. For exemplary methodologies in this regard,see Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes That Confer Resistance to Pests or Disease and That Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant cultivar can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

C. A lectin. See, for example, the disclosure by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor 1), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, a hydroxamic acid, a phenylpropanoidderivative or another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β, lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

R. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes That Confer Resistance to an Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSP) and aroA genes,respectively and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus PAT bar genes), and pyridinoxy or phenoxy propionic acidsand cyclohexones (ACCase inhibitor-encoding genes). See, for example,U.S. Pat. No. 4,940,835 to Shah, et al., which discloses the nucleotidesequence of a form of EPSP which can confer glyphosate resistance. A DNAmolecule encoding a mutant aroA gene can be obtained under ATCCaccession number 39256, and the nucleotide sequence of the mutant geneis disclosed in U.S. Pat. No. 4,769,061 to Comai. European patentapplication No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes which confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a PAT gene is provided in Europeanapplication No. 0 242 246 to Leemans et al. DeGreef et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for PAT activity. Exemplary ofgenes conferring resistance to phenoxy propionic acids and cyclohexones,such as sethoxydim and haloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3genes described by Marshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) or a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

3. Genes That Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.U.S.A. 89:2624 (1992).

B. Decreased phytate content, 1) Introduction of a phytase-encoding genewould enhance breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see Van Hartingsveldt et al., Gene127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene; 2) A gene could be introduced thatreduced phytate content. In maize, this, for example, could beaccomplished, by cloning and then reintroducing DNA associated with thesingle allele which is responsible for maize mutants characterized bylow levels of phytic acid. See Raboy et al., Maydica 35:383 (1990).

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus lichenifonnis α-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), Søogaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme II).

Methods for Rice Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-mediated Transformation—One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch et al., Science227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Miki et al., supra, andMoloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer—Despite the fact the host range forAgrobacterium-mediated transformation is broad, some major cereal cropspecies and gymnosperms have generally been recalcitrant to this mode ofgene transfer, even though some success has recently been achieved inrice and corn. Hiei et al., The Plant Journal 6:271-282 (1994) and U.S.Pat. No. 5,591,616 issued Jan. 7, 1997. Several methods of planttransformation collectively referred to as direct gene transfer havebeen developed as an alternative to Agrobacterium-mediatedtransformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 μm. The expression vectoris introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Sanford et al.,Part. Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299(1988), Klein et al., Bio/Technology 6:559-563 (1988), Sanford, J. C.,Physiol Plant 7:206 (1990), Klein et al., Biotechnology 10:268 (1992).In corn, several target tissues can be bombarded with DNA-coatedmicroprojectiles in order to produce transgenic plants, including, forexample, callus (Type I or Type II), immature embryos, and meristematictissue.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Additionally,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24:51-61 (1994).

Following transformation of rice target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

Singe Gene Conversion

The foregoing methods for transformation would typically be used forproducing a transgenic cultivar. The transgenic cultivar could then becrossed, with another (non-transformed or transformed) cultivar, inorder to produce a new transgenic cultivar. Alternatively, a genetictrait which has been engineered into a particular rice cultivar usingthe foregoing transformation techniques could be moved into anothercultivar using traditional backcrossing techniques that are well knownin the plant breeding arts. For example, a backcrossing approach couldbe used to move an engineered trait from a public, non-elite cultivarinto an elite cultivar, or from a cultivar containing a foreign gene inits genome into a cultivar which does not contain that gene. As usedherein, “crossing” can refer to a simple X by Y cross, or the process ofbackcrossing, depending on the context.

When the term rice plant is used in the context of the presentinvention, this also includes any single gene conversions of thatcultivar. The term single gene converted plant as used herein refers tothose rice plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a cultivar are recovered inaddition to the single gene transferred into the cultivar via thebackcrossing technique. Backcrossing methods can be used with thepresent invention to improve or introduce a characteristic into thecultivar. The term backcrossing as used herein refers to the repeatedcrossing of a hybrid progeny back to one of the parental rice plants,the recurrent parent, for that cultivar, i.e., backcrossing 1, 2, 3, 4,5, 6, 7, 8, 9 or more times to the recurrent parent. The parental riceplant which contributes the gene for the desired characteristic istermed the nonrecurrent or donor parent. This terminology refers to thefact that the nonrecurrent parent is used one time in the backcrossprotocol and therefore does not recur. The parental rice plant to whichthe gene or genes from the nonrecurrent parent are transferred is knownas the recurrent parent as it is used for several rounds in thebackcrossing protocol (Jennings, P. R. et al. Rice Improvement (1979);Mackill D. On your mark, get, select. Rice Today, July-September pp28-29 (2004); Fehr, W. R. et al. Principles of CultivarDevelopment—Theory and Technique (1987)).

In a typical backcross protocol, the original cultivar of interest(recurrent parent) is crossed to a second cultivar (nonrecurrent parent)that carries the single gene of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a rice plant isobtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, in addition to the single transferred gene from thenonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalcultivar. To accomplish this, a single gene of the recurrent cultivar ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original cultivar. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new cultivar but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic, examples of these traits include but are not limited to,male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Some known exceptions to this are the genes for male sterility,some of which are inherited cytoplasmically, but still act as singlegene traits. Several of these single gene traits are described in U.S.Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, the disclosures of whichare specifically hereby incorporated by reference.

Tissue Culture

Further reproduction of the hybrid can occur by tissue culture andregeneration. Tissue culture of various tissues of rice and regenerationof plants therefrom is well known and widely published. For example,reference may be had to Komatsuda, T. et al., Crop Sci. 31:333-337(1991); Stephens, P. A., et al., Theor. Appl. Genet. (1991) 82:633-635;Komatsuda, T. et al., Plant Cell, Tissue and Organ Culture, 28:103-113(1992); Dhir, S. et al., Plant Cell Reports (1992) 11:285-289; Pandey,P. et al., Japan J. Breed. 42:1-5 (1992); and Shetty, K., et al., PlantScience 81:245-251 (1992); as well as U.S. Pat. No. 5,024,944 issuedJun. 18, 1991 to Collins et al., and U.S. Pat. No. 5,008,200 issued Apr.16, 1991 to Ranch et al. Thus, another aspect of this invention is toprovide cells which upon growth and differentiation produce rice plantshaving the physiological and morphological characteristics of ricehybrid XL730.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, glumes,panicles, leaves, stems, roots, root tips, anthers, and the like. Meansfor preparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185; 5,973,234 and5,977,445 describe certain techniques, the disclosures of which areincorporated herein by reference.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cells of tissue culture from which rice plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as pollen, flowers, embryos, ovules,seeds, panicles, glumes, leaves, stems, pistils, anthers and the like.Thus, another aspect of this invention is to provide for cells whichupon growth and differentiation produce a cultivar having essentiallyall of the physiological and morphological characteristics of XL730.

The present invention contemplates a rice plant regenerated from atissue culture of the hybrid rice plant of the present invention. As iswell known in the art, tissue culture of rice can be used for the invitro regeneration of a rice plant. Tissue culture of various tissues ofrice and regeneration of plants therefrom is well known and widelypublished. For example, reference may be had to Chu, Q. R., et al.,(1999) “Use of bridging parents with high anther culturability toimprove plant regeneration and breeding value in rice”, RiceBiotechnology Quarterly 38:25-26; Chu, Q. R., et al., (1998), “A novelplant regeneration medium for rice anther culture of Southern U.S.crosses”, Rice Biotechnology Quarterly 35:15-16; Chu, Q. R., et al.,(1997), “A novel basal medium for embryogenic callus induction ofSouthern US crosses”, Rice Biotechnology Quarterly 32:19-20; and Oono,K., “Broadening the Genetic Variability By Tissue Culture Methods”, Jap.J. Breed. 33 (Suppl.2), 306-307, illus. 1983. Thus, another aspect ofthis invention is to provide cells which upon growth and differentiationproduce rice plants having the physiological and morphologicalcharacteristics of hybrid XL730.

Duncan, et al., Planta 165:322-332 (1985) reflects that 97% of theplants cultured that produced callus were capable of plant regeneration.Subsequent experiments with both cultivars and hybrids produced 91%regenerable callus that produced plants. In a further study in 1988,Songstad, et al., Plant Cell Reports 7:262-265 (1988), reports severalmedia additions that enhance regenerability of callus of two cultivars.Other published reports also indicated that “non-traditional” tissuesare capable of producing somatic embryogenesis and plant regeneration.K. P. Rao et al., Maize Genetics Cooperation Newsletter, 60:64-65(1986), refers to somatic embryogenesis from glume callus cultures andB. V. Conger, et al., Plant Cell Reports, 6:345-347 (1987) indicatessomatic embryogenesis from the tissue cultures of corn leaf segments.Thus, it is clear from the literature that the state of the art is suchthat these methods of obtaining plants are routinely used and have avery high rate of success.

Tissue culture of corn is described in European Patent Application,publication 160,390. Corn tissue culture procedures are also describedin Green and Rhodes, “Plant Regeneration in Tissue Culture of Maize,”Maize for Biological Research (Plant Molecular Biology Association,Charlottesville, Va. 367-372, (1982)) and in Duncan et al., “TheProduction of Callus Capable of Plant Regeneration from Immature Embryosof Numerous Zea Mays Genotypes”, 165 Planta 322:332 (1985). Thus,another aspect of this invention is to provide cells which upon growthand differentiation produce rice plants having the physiological andmorphological characteristics of rice hybrid XL730.

Additional Breeding Methods

The utility of rice hybrid XL730 also extends to crosses with otherspecies. Commonly, suitable species will be of the family Graminaceae,and especially of the genera Zea, Tripsacum, Croix, Schlerachne,Polytoca, Chionachne, and Trilobachne, of the tribe Maydeae.

This invention also is directed to methods for producing a rice plant bycrossing a first parent rice plant with a second parent rice plantwherein the first or second parent rice plant is a rice plant of thehybrid XL730. Further, both first and second parent rice plants can comefrom the rice hybrid XL730. Thus, any such methods using the rice hybridXL730 are part of this invention: selfing, backcrosses, hybridproduction, crosses to populations, and the like. All plants producedusing rice hybrid XL730 as a parent are within the scope of thisinvention, including those developed from varieties derived from ricehybrid XL730. Advantageously, the rice hybrid of the present inventioncould be used in crosses with other, different, rice plants to producethe first generation (F₁) rice hybrid seeds and plants with superiorcharacteristics. One or both parents of the hybrid of the invention canalso be used for transformation where exogenous genes are introduced andexpressed by one or both of the parents of the invention. Geneticvariants created either through traditional breeding methods using oneor both of the parents of XL730 or through transformation of one or bothof the parents of XL730 by any of a number of protocols known to thoseof skill in the art are intended to be within the scope of thisinvention.

The following describes breeding methods that may be used with hybridXL730 or with one or both of the parents of hybrid XL730 in thedevelopment of further rice plants. One such embodiment is a method fordeveloping an XL730-derived progeny rice plant in a rice plant breedingprogram comprising: obtaining the rice plant, or a part thereof, of riceline XL730, utilizing said plant or plant part as a source of breedingmaterial and selecting an XL730 progeny plant with molecular markers incommon with XL730 and/or with morphological and/or physiologicalcharacteristics selected from the characteristics listed in Tables 1, 2,3, or 4. The same method may be used with one or both of the parents ofXL730. Breeding steps that may be used in the rice plant breedingprogram include pedigree breeding, back crossing, mutation breeding, andrecurrent selection. In conjunction with these steps, techniques such asRFLP-enhanced selection, genetic marker enhanced selection (for exampleSSR markers) and the making of double haploids may be utilized.

Another method involves producing a population of XL730 progeny riceplants, comprising crossing rice hybrid XL730 with another rice plant,thereby producing a population of rice plants, which, on average, derive50% of their alleles from rice hybrid XL730. A plant of this populationmay be selected and repeatedly selfed or sibbed with a rice cultivarresulting from these successive filial generations. One embodiment ofthis invention is the rice cultivar produced by this method and that hasobtained at least 50% of its alleles from hybrid XL730. The same methodmay be used with one or both of the parents of XL730.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, p 261-286 (1987). Thus the invention includes rice hybridXL730 progeny rice plants comprising a combination of at least two XL730traits selected from the group consisting of those listed in Tables 1,2, 3, and 4 or the XL730 combination of traits listed in the Summary ofthe Invention, so that said progeny rice plant is not significantlydifferent for said traits than rice hybrid XL730. Using techniquesdescribed herein, molecular markers may be used to identify said progenyplant as a XL730 progeny plant. Mean trait values may be used todetermine whether trait differences are significant, and preferably thetraits are measured on plants grown under the same environmentalconditions. Once such a variety is developed its value is substantialsince it is important to advance the germplasm base as a whole in orderto maintain or improve traits such as yield, disease resistance, pestresistance, and plant performance in extreme environmental conditions.

Progeny of rice hybrid XL730 may also be characterized through theirfilial relationship with rice hybrid XL730, as for example, being withina certain number of breeding crosses of rice hybrid XL730. A breedingcross is a cross made to introduce new genetics into the progeny, and isdistinguished from a cross, such as a self or a sib cross, made toselect among existing genetic alleles. The lower the number of breedingcrosses in the pedigree, the closer the relationship between rice hybridXL730 and its progeny. For example, progeny produced by the methodsdescribed herein may be within 1, 2, 3, 4 or 5 breeding crosses of ricehybrid XL730.

The seed of rice hybrid XL730, the plant produced from the hybrid seed,the hybrid rice plant produced from the crossing of the hybrid, andvarious parts of the hybrid rice plant and transgenic versions of theforegoing, can be utilized for human food, livestock feed, and as a rawmaterial in industry.

Tables

The following tables present data on the traits and characteristics ofrice hybrid XL730 as compared with CL 161, a commonly grown CLEARFIELDrice variety in the U.S. The data were collected from multiple locationsand repeated trials. In the following tables, probability figuresindicate the probability associated with a paired Student's t-Test usedto determine whether two samples are likely to have come from the sametwo underlying populations that have the same mean.

The data presented in Table 2 compare yield, plant height, maturity,lodging and milling yields of CL161 versus the hybrid of the presentinvention produced by crossing 053004 with 054001. As shown in Table 2,rice hybrid XL730 unexpectedly has significantly higher yield thanCL161, is significantly taller and flowers earlier than CL161.Additionally, rice hybrid XL730 has significantly lower whole millingyields and higher lodging than CL161.

In Table 2 below, column 2 shows the yield in kilograms per hectare,column 3 shows the plant height in centimeters, column 4 shows thenumber of days to 50% flowering, column 5 shows the percent lodging,column 6 shows the total milling percent and column 7 shows the wholemilling percent. The number of different locations over which the datawere collected is shown in row 3. TABLE 2 Plant Yield Height Days to 50%Total Whole (kg/ha) (cm) Flowering Lodging % Milling % Milling % XL73012,191 111 82 29 72.0 61.3 CL 161 9,497 96 84 12 71.6 65.9 Locations 147 7 4 13 13 Difference 2694 15 −2 17 0.4 −4.6 Probability 0.000 0.0000.006 0.040 0.058 0.000

The data presented in Table 3 compare the basic quality characteristicsof rice hybrid XL730 and CL161. Unexpectedly, rice hybrid XL730 hadsignificantly lower amylose, alkali spreading value and grain length towidth ratio than CL161. Also, the grain length, width, and chalk percentof hybrid XL730 are significantly greater than those of CL161.

In Table 3 below, column 2 shows the percent amylose, column 3 shows thealkali spreading value, column 4 shows the grain length in millimeters,column 5 shows the grain width in millimeters, column 6 shows the grainlength to width ratio and column 7 shows the percent grain chalk. Thenumber of different locations over which the data were collected isshown in row 3. TABLE 3 L/W Amylose ASV Length Width Ratio Chalk XL73019.7 3.9 6.68 2.18 3.07 15 CL 161 21.0 4.7 6.61 2.07 3.20 7 Locations 1112 10 10 10 6 Difference −1.3 −0.8 0.07 0.11 −0.13 19 Probability 0.0010.001 0.014 0.000 0.000 0.034

Table 4 compares starch characteristics between rice hybrid XL730 and CL161. Rice hybrid XL730 has a higher peak viscosity, paste temperature,paste time and breakdown than CL161. For peak time, final viscosity,setback and consistency rice hybrid XL730 has lower values than CL161.

In Table 4 below, column 2 shows the peak viscosity, column 3 shows thepeak time, column 4 shows the trough, column 5 shows the trough time,column 6 shows the paste temperature in degrees C, column 7 shows thepaste time, column 8 shows the final viscosity, column 9 shows thebreakdown, column 10 shows the setback and column 11 shows theconsistency of the starch. TABLE 4 Paste Peak Peak Trough Temp PasteFinal Viscosity Time Trough Time (° C.) Time Viscosity Breakdown SetbackConsistency XL730 278 6.50 131 9.59 74.7 3.69 253 147 −119 122 CL 161244 6.59 128 9.56 74.0 3.54 261 116 −56 132 Observations 16 16 16 16 1616 16 16 4 16 Difference 34 −0.09 3 0.03 0.7 0.15 −7 31 −164 −10Probability 0.000 0.018 0.159 0.198 0.003 0.009 0.014 0.000 0.001 0.001

Deposit Information

Deposits of seed of each of the parents of this invention are maintainedby RiceTec, Inc., 1925 FM 2917, Alvin, Tex. 77511. Access to thesedeposits will be available during the pendency of this application topersons determined by the Commissioner of Patents and Trademarks to beentitled thereto under 37 CFR 1.14 and 35 USC 122. Upon allowance of anyclaims in this application, all restrictions on the availability to thepublic of the parents of the hybrid will be irrevocably removed byaffording access to deposits of at least 2,500 seeds of each of the sameparents of the hybrid with the American Type Culture Collection,Manassas, Va.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

1. A hybrid rice seed designated XL730 having rice cultivar 054001 as afirst parental line, wherein a representative sample of seed of cultivar054001 was deposited under ATCC Accession No. ______, and having ricecultivar 053004 as a second parental line, wherein a representativesample of seed of cultivar 053004 was deposited under ATCC Accession No.PTA ______.
 2. A rice plant, or a part thereof, produced by growing theseed of claim
 1. 3. Pollen of the plant of claim
 2. 4. An ovule of theplant of claim
 2. 5. A rice plant, or a part thereof, having all of thephysiological and morphological characteristics of the rice plant ofclaim
 2. 6. A tissue culture of cells produced from the rice plant ofclaim 2, wherein said cells of the tissue culture are produced from aplant part selected from the group consisting of leaves, pollen,embryos, cotyledon, hypocotyl, meristematic cells, roots, root tips,pistils, anthers, flowers, stems, glumes and panicles.
 7. A protoplastproduced from the plant of claim
 2. 8. A rice plant regenerated from thetissue culture of claim 6, wherein the plant has all the morphologicaland physiological characteristics of hybrid XL730.
 9. A method forproducing a rice seed, wherein the method comprises crossing the plantof claim 2 with a different rice plant and harvesting the resultanthybrid rice seed.
 10. A hybrid rice seed produced by the method of claim9.
 11. A hybrid rice plant, or a part thereof, produced by growing saidhybrid seed of claim
 10. 12. A method of producing an herbicideresistant rice plant wherein the method comprises transforming one orboth of the parents of the rice plant of claim 2 with a transgenewherein the transgene confers resistance to an herbicide selected fromthe group consisting of imidazolinone, sulfonylurea, glyphosate,glufosinate, L-phosphinothricin, triazine and benzonitrile.
 13. Anherbicide resistant rice plant produced by the method of claim
 12. 14. Amethod of producing an insect resistant rice plant wherein the methodcomprises transforming one or both of the parents of the rice plant ofclaim 2 with a transgene that confers insect resistance.
 15. An insectresistant rice plant produced by the method of claim
 14. 16. The riceplant of claim 15, wherein the transgene encodes a Bacillusthuringiensis endotoxin.
 17. A method of producing a disease resistantrice plant wherein the method comprises transforming one or both parentsof the rice plant of claim 2 with a transgene that confers diseaseresistance.
 18. A disease resistant rice plant produced by the method ofclaim
 17. 19. A method of producing a rice plant with modified fattyacid metabolism or modified carbohydrate metabolism wherein the methodcomprises transforming one or both parents of the rice plant of claim 2with a transgene encoding a protein selected from the group consistingof fructosyltransferase, levansucrase, alpha-amylase, invertase andstarch branching enzyme or encoding an antisense of stearyl-ACPdesaturase.
 20. A rice plant having modified fatty acid metabolism ormodified carbohydrate metabolism produced by the method of claim 19.